Plant biomass or lignocellulose is the most abundant renewable energy source on earth. From the viewpoints of global environmental conservation and fossil fuel depletion, biorefinery using plant biomass as a biofuel or a raw material of chemical products such as ethanol has attracted attention. The main component in the dry weight of plant biomass is lignocellulose composed of polysaccharides, such as celluloses and hemicelluloses, and lignin. For example, polysaccharides are hydrolyzed into monosaccharides such as glucose and xylose by glycoside hydrolases, and are then used as a biofuel or a raw material of chemical products.
Lignocellulose having a complex structure is persistent and is difficult to degrade or hydrolyze with a single glycoside hydrolase enzyme. For the complete degradation of lignocellulose, in general, three types of enzymes, i.e., an endoglucanase (cellulase or endo-1,4-β-D-glucanase, EC 3.2.1.4), an exo-type cellobiohydrolase (1,4-β-cellobiosidase or cellobiohydrolase, EC 3.2.1.91, EC 3.2.1.176), and a β-glucosidase (EC 3.2.1.21) are required. In addition, it is considered that an appropriate formulation of multiple enzymes is necessary, including a xylanase (endo-1,4-β-xylanase, EC 3.2.1.8) which is a hemicellulase and other plant cell wall degrading enzymes.
When cellulose is subjected to hydrolysis by cellobiohydrolase, cellobiose which is a disaccharide is mainly produced. β-glucosidase hydrolyzes this cellobiose into glucose, which is a monosaccharide, and is therefore one of the essential enzymes for degrading lignocellulose ultimately to monosaccharides.
In conventional bioethanol production using lignocellulose as a starting resource, hydrolysis processes using high solid loading (30 to 60% solid loading) have been tested with the aim of achieving a more energy-efficient conversion to ethanol. However, in this type of lignocellulose enzymatic hydrolysis using high solid loading, the viscosity of the biomass slurry (hydrolyzed biomass solution) is high, and the hydrolysis reaction of the lignocellulose tends to proceed poorly. Accordingly, by using a thermostable enzyme and performing the enzymatic hydrolysis treatment at a high temperature of 80° C. or higher, for example, the rate of the hydrolysis reaction can be increased, and the viscosity of the biomass slurry can be reduced, which is expected to enable a shortening of the hydrolysis reaction time and a reduction in the amount of enzyme required. As a result, for all of the various glycoside hydrolases, the development of enzymes having superior thermostability is very desirable.
Many thermostable enzymes have been obtained by cloning genes from thermophilic microorganisms that exist in high-temperature environments, determining the DNA sequence, and then expressing the DNA using E. coli or filamentous fungi or the like. For example, a thermostable β-glucosidase (with an optimum temperature of 70° C. and an optimum pH of 3.5 to 4.0) derived from a filamentous fungus Acremonium cellulolyticus has been disclosed in Patent Document 1. Three types of thermostable β-glucosidases (with an optimum temperature of 55° C. and an optimum pH of 4.5 to 5.1) derived from Acremonium cellulolyticus have been disclosed in Patent Document 2. A thermostable β-glucosidase (with an optimum temperature of 80° C. and an optimum pH of 5 to 6) derived from a Thermoanaerobactor species has been disclosed in Patent Document 3. A thermostable β-glucosidase (with an optimum temperature of 80° C. and an optimum pH of 4.6) derived from Thermoascas auranticus has been disclosed in Non-Patent Document 1. A thermostable β-glucosidase (with an optimum temperature of 90° C. and an optimum pH of 6 to 7) derived from Fervidobacterium islandicum has been disclosed in Non-Patent Document 2.
In addition, since β-glucosidases generally have characteristics so that their activities are inhibited by glucose which is their decomposition product, although the development of β-glucosidases that are less susceptible to glucose has been advanced, those having high thermostability are not available (for example, see Patent Document 4, and Non-Patent Documents 3 and 4).